Retinoblastoma (RB) is the most common intraocular malignancy of childhood. Enucleation (eye removal) is effective treatment, but vision-preserving approaches, such as radiation and chemotherapy have become the standard of care. Despite these advances in treatment, many survivors of RB develop metastatic disease or secondary tumors. We propose that there are subpopulation(s) of RB cells that escape standard treatments to form new tumors. The cancer stem cell theory suggests that only a small percentage of cells within a tumor are capable of driving tumor growth and metastasis, whereas the majority of a tumor is actually made up of non-tumorigenic cells. Tumors recur because a small number of chemo-resistant tumor-initiating cells survive to repopulate the tumor. At present, there is a gap in knowledge regarding the existence and potential role of tumor-initiating cells in RB. Our central hypothesis is that RB tumors contain subpopulation(s) of tumor-initiating, chemo-resistant "cancer stem cells" that promote tumor progression. We base this hypothesis on our previous findings of heterogeneity in both RB tumors and cell lines, in which subpopulations of RB cells display a number of stem cell characteristics: the ability to exclude Hoechst 33342 dye as a "side population" (SP), expression of ABGC2 (a stem cell/chemo drug resistance marker), as well as immunoreactivity to human stem cell markers such as Oct3/4, Nanog and Musashi-1 (genes of self-renewal and pluripotency). Our overall goal is to isolate and characterize subpopulations of tumor-initiating RB cells via the following methods: With access to live human RB cells from over 200 tumors, we will identify potential tumor- initiating cells using fluorescence activated cell sorting based the following criteria: 1) the expression of cell surface markers that are expressed by tumor-initiating cells in other tissues;such as ABCG2, 2) the ability to efflux the fluorescent dye, Hoechst 33342 and 3) the expression of genes involved in stem cell renewal/pluripotency such as Oct3/4, Nanog and Musashi-1. We will assess specific features of these subpopulations that correspond with functional attributes of cancer stem cells, such as self-renewal and slow cell cycling. We will then compare the relative ability of cell populations with and without these properties to form tumors in immunodeficient NOD-SCID mice. We will identify potential new therapeutic targets in RB by comparing gene expression profiles of cell populations enriched for tumor-initiating cells to their non-tumor forming counterparts. Results from these studies will define the tumor-initiating phenotype(s) in RB, and assess their potential as novel therapeutic targets in retinoblastoma, as well as other types of malignancies. PUBLIC HEALTH RELEVANCE: Retinoblastoma, the most common intraocular tumor of childhood, appears to contain a small population of cells that behave as "cancer stem cells". The goal of our project is to identify and target cancer stem cells in retinoblastoma to show that they are primarily responsible for tumor progression and resistance to chemotherapy drugs. Results from our study will lead to new treatment strategies for retinoblastoma and other tumors that contain these small populations of cancer stem cells.